Spatial pattern separation (OPS) task
The spatial pattern separation (OPS) task (based on van Goethem et al., 2018) was performed to evaluate the animals’ capacity to discriminate minimal changes in the spatial position of identical objects in a familiar arena. To reduce the animals’ stress from the experimental manipulation, the experimenter manipulated the rats twice per day (2-3 min) for five consecutive days before starting the behavioral evaluations. A circular arena (40 cm high and 83 cm in diameter, gray wall) was placed on a black acrylic platform inside an experimental room with red light. On this platform, a series of reference points’ denominated positions (P) were designated as P1 to P5, with 6 cm between the Ps. P1 was aligned at the center of the platform, both left and right, and then P2 to P5 were designated both upward and downward, as depicted in Figure 9a.
The OPS task consisted of two trials (3 min duration each): the learning trial (T1) and the discrimination trial (T2). Before T1 in each evaluation, a habituation phase was carried out, during which the animal was exposed to the empty arena for 5 min. In T1, the animal was introduced to the arena with two identical objects placed in P1 from the left and right of the platform. One hour later, in T2, the animal was re-exposed to the same objects, but one object was displaced to a new position; this then carried on to include all other positions (P2-P5). Object exploration was considered for analysis when the animal explored an object with its nose, with a minimal distance of 2 cm. From the time of exploration for each object, we calculated the discrimination index (DI), a quantitative measurement of the animals’ preference for exploring the displaced object over the stationary object, using the following formula:
\begin{equation} DI=\frac{\left(displaced\ objects\ exploration\ time-stationary\ object^{\prime}\ s\ exploration\ time\right)}{two\ objects\ total\ exploration\ time}\nonumber \\ \end{equation}
To estimate the DI, a minimum exploration time of 7 and 10 s was required for T1 and T2, respectively (van Goethem et al., 2018). Likewise, the performance of each animal for all positions was determined. Therefore, the animals were exposed 5 times to the OPS task with an interval of 2 days and different par objects, which prevented familiarization (van Goethem et al., 2018). The order of evaluation of object positions was random: while one animal began with P1 (without displacement of objects), another animal began with P5 (maximal displacement of one object). After each trial, the platform and the objects were cleaned with ethanol (70%) to eliminate odor residues. The first evaluation of the OPS task was carried out in animals of P30.